Arimoclomol for treating glucocerebrosidase associated disorders

ABSTRACT

The present invention relates to an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximioyl chloride, its stereoisomers and the acid addition salts thereof (arimoclomol), for use in a method of treating glucocerebrosidase associated disorders.

TECHNICAL FIELD

The present invention relates to an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof(arimoclomol), for use in a method of treating a glucocerebrosidase(GBA)-associated disorder other than Gaucher's disease (GD), includingGBA-associated alpha-synucleinopathies such as GBA-associatedParkinson's disease (PD), GBA-associated dementia with Lewi bodies (DLB)and GBA-associated multiple system atrophy (MSA).

BACKGROUND

Gaucher's disease (GD) is the most common of the lysosomal storagediseases characterized by an accumulation of glucocerebrosides. It is aform of sphingolipidosis as it involves dysfunctional metabolism ofsphingolipids. To date, up to 300 mutations in the GBA gene are knownand linked to Gaucher disease. GBA mutations can be categorized as mild(causing GD type I, nonneuronopathic) or severe (causing GD types II andIII). Homozygous GBA mutations as well as compound heterozygousmutations cause GD. A few common mutations predominate, the mostprevalent for GD Type I being a missense mutation resulting in thesubstitution of a serine for asparagine at amino acid residue 370(N370S), and the most prevalent for Type II and III being L444P (Codonsare numbered from the first codon of the mature protein i.e. without thesignal peptide).

Many of these mutations are also found in patients with Parkinson'sdisease (PD). Heterozygous mutations as found in GBA mutation carriers(having one mutated GBA gene) are found to predispose for development ofParkinson's disease (Gan-Or et al., Neurology, 2015). Mutations in GBAare now considered one of the main genetic risk factors for Parkinson'sdisease. It has been estimated that at least 8% of patients withParkinson's disease have mutations in the GBA gene, both mild and severeGBA mutations, including L444P heterozygotes. Also secondarydeficiencies of GBA activity may be linked to Parkinson's disease.

The primary pathology leading from GBA deficiency to Parkinson's diseaseis not clarified, but pre-clinical experiments suggest an inverserelationship to α-synuclein. Carriers of GBA gene mutations appear alsoto have an increased risk of developing dementia with Lewy bodies (DLB)and possibly multiple system atrophy (MSA), providing a link between GBAdeficiency and at least some of the alpha-synucleinopathies.

WO 2014/071282 discloses a recombinant self-complementaryadeno-associated viral vector encoding human glucocerebrosidase(AAV-GBAI) in models to support glucocerebrosidase augmentationtherapies for PD and related synucleinopathies and tauopathies.

WO 2013/148333 discloses salicylic acid derivatives asglucocerebrosidase activators for treating Gaucher's disease andinhibiting the onset of Gaucher's disease symptoms in a patient having aGBA gene mutation and for treating Parkinson's disease.

WO 2009/155936 discloses heat shock protein 70 and inducers thereof fortreating lysosomal storage diseases, including Gaucher's disease.

WO 2005/041965 discloses use of the heat shock inducer arimoclomol forprotecting neurons in neurodegenerative diseases, including Parkinson'sdisease.

SUMMARY

Arimoclomol is a heat shock protein amplifier currently under evaluationin the treatment of paediatric lysosomal storage disorders andamyotrophic lateral sclerosis (ALS).

The present inventors have now found that arimoclomol increases GBAlevels and increases GBA activity, not only in GBA homozygotes(presenting with Gaucher's disease and markedly reduced GBA activity),but also in mutant GBA heterozygotes (carriers). Specifically,arimoclomol increases GBA activity in GBA homozygotes (Gaucher patient)to clinically unaffected activity level. Furthermore, arimoclomolincreases GBA activity in GBA heterozygotes (clinically unaffected), andincreases GBA enzyme amount (total level and matured/post-ER GBA).

The present inventors also show herein that arimoclomol increases GBAactivity in Parkinson's disease patients with mutated GBA alleles(heterozygous or homozygous, clinically unaffected re Gaucher'sdisease).

It is an aspect to provide an active pharmaceutical ingredient selectedfromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride (arimoclomol), its stereoisomers and the acid addition saltsthereof, for use in a method of treating a glucocerebrosidase(GBA)-associated disorder.

In one embodiment said GBA-associated disorder is associated withreduced GBA enzyme levels and/or reduced GBA enzyme activity. In oneembodiment said GBA-associated disorder is associated with one or moreGBA gene mutations, including heterozygous and homozygous GBA genemutations.

In one embodiment said GBA-associated disorder is a GBA-associatedalpha-synucleinopathy, such as selected from the group consisting ofGBA-associated Parkinson's disease (PD), GBA-associated dementia withLewi bodies (DLB) and GBA-associated multiple system atrophy (MSA).

In one embodiment said GBA-associated Parkinson's disease is associatedwith a genetically high-risk Parkinson's disease GBA genotype.

Also provided is an active pharmaceutical ingredient selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of increasing GBA levels and/or

GBA activity.

DESCRIPTION OF DRAWINGS

FIG. 1: Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) inprimary cells (human fibroblasts) from individual with a heterozygousGBA allele containing the L444P, A456P, V460V mutations in cis (carrier,clinically unaffected re. Gaucher's disease). See Example 1.

FIG. 2: Arimoclomol-induced dose-dependent increase in GBA enzyme amountin primary cells (human fibroblasts) from individual with a heterozygousGBA allele containing the L444P, A456P, V460V mutations in cis (carrier,not affected with Gaucher's disease). See Example 1.

FIG. 3: Arimoclomol-induced dose-dependent increase in GBA activity inL444P/L444P, A456P, V460V Gaucher TII patient. Level increased toclinically unaffected activity level (dashed line). See Example 2.

FIG. 4: Arimoclomol-induced dose-dependent increase in GBA activity inL444P, A456P, V460V Heterozygote (carrier; clinically unaffected parentof Gaucher disease patient, genetically high-risk Parkinson's diseasegenotype). Level increased by more than 2-fold. See Example 2.

FIG. 5: Arimoclomol-induced dose-dependent increase in GBA activity inprimary cells from Gaucher disease patients of type I (N370S/V394L andN370S/1-BP ins 84G), type II (E326K, L444P/E326K, L444P and G325R/C342Gand P415R/L444P) or type Ill (L444P/L444P). See Example 3.

FIG. 6: Arimoclomol-induced dose-dependent increase in GBA activity inprimary cells from Parkinson Disease patient with a heterozygous GBAallele containing the N370S mutation (N370S/+). See Example 4.

FIG. 7. Arimoclomol-induced dose-dependent increase in GBA activity inhuman fibroblasts from non-symptomatic healthy individuals with no GBAmutation (+/+). See Example 5.

FIG. 8: Arimoclomol-induced increase in labeling of active GBA by ME569in primary cells from Gaucher disease patients of type I (N370S/V394L),type II (G325R/C342G) and type III (L444P/L444P). See Example 6.

FIG. 9: Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) inprimary cells from Gaucher disease patient of type I (N370S/V394L).Vinculin was used as loading control. See Example 7.

FIG. 10: Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP)in primary cells from Gaucher disease patient of type I (N370S/1-BP ins84G). RPA was used as loading control. See Example 7.

FIG. 11: Arimoclomol-induced dose-dependent increase in GBA proteinlevel in primary cells from Gaucher disease patient of type I(N370S/1-BP ins 84G). RPA was used as loading control. See Example 7.

FIG. 12: Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP)in primary cells from Gaucher disease patient of type II (L444P/P415R).RPA was used as loading control. See Example 8.

FIG. 13: Arimoclomol-induced increase in ER Hsp70 (BiP) in primary cellsfrom Gaucher disease patient of type II (G325R/C342G). Vinculin was usedas loading control. See Example 8.

FIG. 14: Arimoclomol-induced dose-dependent increase in GBA proteinlevel in primary cells from Gaucher disease patient of type II(L444P/P415R). Vinculin was used as loading control. See Example 8.

FIG. 15: Arimoclomol-induced dose-dependent increase in GBA proteinlevel in primary cells from Gaucher disease patient of type II(G325R/C342G). RPA was used as loading control. See Example 8.

FIG. 16: Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP)in primary cells from Gaucher disease patient of type III (L444P/L444P).Vinculin was used as loading control. See Example 9.

FIG. 17: Arimoclomol-induced dose-dependent increase in GBA proteinlevel in primary cells from Gaucher disease patient of type III(L444P/L444P). RPA was used as loading control. See Example 9.

FIG. 18: Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP)in primary cells from a PD-GBA (N370S/N370S) individual. Vinculin wasused as loading control. See Example 10.

FIG. 19: Arimoclomol does not affect neuronal differentiation of MASCsfrom GD individuals with the indicated GBA mutations. Cells were eithertreated with mock (PBS) or 400 μM arimoclomol (Ari) for 9 days. Theexpression of the neuronal markers Tubulin beta 3 (FIG. 19 A) and NeuN(FIG. 19 B) was evaluated by immunostaining. See Example 11.

FIG. 20: Arimoclomol-induced increase in GBA activity in primaryneronal-like cells from GD individuals with the indicated GBA mutations.Skin-derived fibroblasts from individual with GDTIII (L444P/L444P) wereincluded as control. Cells were either treated with mock (PBS) or 400 μMarimoclomol (Ari). See Example 11.

DETAILED DESCRIPTION

Beta-glucocerebrosidase or glucocerebrosidase (UniProt entry P04062,GLCM_HUMAN, also called glucosylceramidase, acid beta-glucosidase,D-glucosyl-N-acylsphingosine glucohydrolase, GCase or GBA) is an enzymewith glucosylceramidase activity that cleaves, by hydrolysis, thebeta-glucosidic linkage of glucocerebroside, an intermediate inglycolipid metabolism:

D-glucosyl-N-acylsphingosine+H₂O=D-glucose+N-acylsphingosine.

GBA requires saposin C and anionic phospholipids for activity. It islocalized in the lysosome. It is encoded by the GBA gene (official name:glucosidase, beta, acid; Gene/Locus MIM number 606463; EC 3.2.1.45).Alternative splicing results in multiple transcript variants.

Mutations in the GBA gene, which encodes the lysosomal enzyme that isdeficient in Gaucher's disease, are important and common risk factorsfor Parkinson's disease and related disorders. This association wasfirst recognised in the clinic, where parkinsonism was noted, albeitrarely, in patients with Gaucher's disease and more frequently inrelatives who were obligate carriers (an individual who may beclinically unaffected but who must carry a gene mutation based onanalysis of the family history).

GBA gene mutations are continuously updated in the LOVD CCHMC MolecularGenetics Laboratory Mutation Database, Gaucher Disease; glucosidase,beta, acid (GBA) athttps://research.cchmc.org/LOVD2/home.php?select_db=GBA.

Subsequently, findings from large studies showed that patients withParkinson's disease and associated Lewy body disorders had an increasedfrequency of GBA mutations when compared with control individuals.Patients with GBA-associated parkinsonism exhibit varying parkinsonianphenotypes but tend to have an earlier age of onset and more associatedcognitive changes than patients with parkinsonism without GBA mutations.Hypotheses proposed to explain this association include again-of-function due to mutations in glucocerebrosidase that promotesα-synuclein aggregation; substrate accumulation due to enzymaticloss-of-function, which affects α-synuclein processing and clearance;and a bidirectional feedback loop.

Alpha-synuclein is a synuclein protein of unknown function primarilyfound in neural tissue. It can aggregate to form insoluble fibrils inpathological conditions characterized by Lewy bodies, such asParkinson's disease, dementia with Lewy bodies, and multiple systematrophy. Alpha-synuclein is the primary structural component of Lewybody fibrils.

Arimoclomol is a small-molecule inducer of the heat shock proteinsincluding Hsp70. It is currently being investigated for treatment ofamyotrophic lateral sclerosis (ALS) and the lysosomal storage disorderNiemann-Pick disease type C. Induction of the heat shock proteinsincluding Hsp70 protects lysosomal membranes and increases activity oflysosomal enzymes responsible for degradation of lysosomal substrate.

The present inventors show herein that arimoclomol increases GBAactivity in cells from a patient with Gaucher's disease type III (e.g.L444P/L444P) to clinically unaffected activity levels (in some instancessame levels as GBA mutation carriers). Also shown herein is thatarimoclomol surprisingly increases GBA activity in cells from a GBAmutation carrier (e.g. L444P heterozygous) more than 2-fold ofclinically unaffected activity levels. Furthermore, arimoclomolincreases N370S GBA activity in cells from a PD patient. Thus, GBAactivity—and levels—can be increased also in cells from mutant GBAheterozygotes (carriers), and in cells from mutant GBA homozygotes whoare clinically unaffected re Gaucher's disease.

Arimoclomol-induced increase in GBA levels and/or activity may thusprovide useful for treating a range of proteinopathic disorders whereinGBA levels and/or activity is compromised.

Arimoclomol is defined herein as an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof.

Provided herewith is arimoclomol for use in the treatment of GBAdeficiencies. In one embodiment said GBA deficiency does not includeGaucher's disease (GD) per se/as such.

Provided herewith is arimoclomol for use in the treatment of aglucocerebrosidase (GBA)-associated disorder other than Gaucher'sdisease (GD).

In one embodiment said treatment is prophylactic, curative orameliorating. In one particular embodiment, said treatment isprophylactic. In another embodiment, said treatment is curative. In afurther embodiment, said treatment is ameliorating.

Also provided herewith is use of arimoclomol for the manufacture of amedicament for the treatment of a glucocerebrosidase (GBA)-associateddisorder other than Gaucher's disease (GD).

Also provided herewith is a method of treating a glucocerebrosidase(GBA)-associated disorder other than Gaucher's disease (GD), said methodcomprising administering an effective amount of arimoclomol to anindividual in need thereof.

The term “Individual” or “subject” refers to vertebrates, in particulara member of a mammalian species, preferably primates including humans.In a preferred embodiment, an individual as used herein is a humanbeing, male or female, of any age.

An “individual in need thereof” refers to an individual who may benefitfrom the present invention. In one embodiment, said individual in needthereof is a diseased individual, wherein said disease is aGBA-associated disorder.

GBA-Associated Disorders

In one embodiment there is provided a compound selected from the groupconsisting of(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate;(−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate;(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate; and(−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate, for use in the treatment of a GBA-associated disorder.

Reference to arimoclomol, as defined herein, for use in the treatment ofa GBA-associated disorder encompasses any one of the below conditions.

A GBA-associated disorder as defined herein may refer to any disorderwhich has an association with GBA levels and/or GBA activity. Thus,reduced levels and/or reduced activity of GBA is associated with aGBA-associated disorder as defined herein. Associated with in oneembodiment means predisposes for (or increases risk of developing; orpresenting with).

In one embodiment the GBA-associated disorder is not Gaucher's disease.In one embodiment the GBA-associated disorder is not Gaucher's diseasetype I. In one embodiment the GBA-associated disorder is not Gaucher'sdisease type II. In one embodiment the GBA-associated disorder is notGaucher's disease type III. In one embodiment the GBA-associateddisorder is not Gaucher's disease types II or III.

In one embodiment the GBA-associated disorder is associated with reducedGBA enzyme levels.

In one embodiment the GBA-associated disorder is associated with reducedGBA enzyme activity.

Reduced GBA enzyme levels and/or GBA activity may also be defined asimpaired GBA enzyme levels and/or GBA activity; insufficient GBA enzymelevels and/or GBA activity; or deficient GBA enzyme levels and/or GBAactivity.

In one embodiment the GBA-associated disorder is referred to as aGBA-deficiency.

In one embodiment the GBA-associated disorder has a GBA activity and/orenzyme level which is reduced yet sufficient to remain clinicallyunaffected with respect to Gaucher's disease (i.e. does not have and isnot diagnosed with Gaucher's disease). In one embodiment theGBA-associated disorder has a GBA activity and/or enzyme level which isreduced compared to wild type activity levels.

In one embodiment the GBA-associated disorder is associated with one ormore individual GBA gene mutations. In one embodiment the GBA-associateddisorder is an individual having one or more GBA gene mutations whoremain clinically unaffected re Gaucher's disease.

In one embodiment the GBA-associated disorder is associated with one ormore mild GBA gene mutations (associated with GD type I; TI).

In another embodiment the GBA-associated disorder is associated with oneor more severe GBA gene mutations (associated with GD type II; TII, andGD type III; TIII).

In one embodiment the GBA-associated disorder is associated with one ormore heterozygous GBA gene mutations, wherein said heterozygous GBA genemutations do not cause or result in the development of Gaucher'sdisease.

In one embodiment the GBA-associated disorder is an individual havingone or more heterozygous GBA gene mutations who remain clinicallyunaffected re Gaucher's disease.

In one embodiment the GBA-associated disorder is associated with one ormore homozygous GBA gene mutations and/or compound heterozygous GBA genemutations, wherein said GBA gene mutations do not cause or result in thedevelopment of Gaucher's disease.

In one embodiment the GBA-associated disorder is an individual havingone or more homozygous and/or compound heterozygous GBA gene mutationswho remain clinically unaffected re Gaucher's disease.

Specific mutations in the GBA gene that may affect the activity of theGBA protein include L444P, D409H, D409V, E235A, E340A, E326K, N370S,N370S/1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*,F213I, N188S and IVS2+1G>A/N188S.

In one embodiment, the GBA-associated disorder is associated with (orcomprises, presents with) one or more mutations in the GBA gene selectedfrom the group consisting of L444P, D409H, D409V, E235A, E340A, E326K,N370S, N370S/1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415RY133*, F213I, N188S and IVS2+1G>A/N188S. Said one or more mutations inthe GBA gene can be heterozygous, compound heterozygous or homozygousmutations.

In one embodiment the GBA-associated disorder is an individual havingone or more GBA gene mutations selected from the group consisting ofL444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S/1-BP ins 84G,V394L, A456P, V460V, C342G, G325R, P415R Y133*, F213I, N188S andIVS2+1G>A/N188S who remain clinically unaffected re Gaucher's disease.Said one or more mutations in the GBA gene can be heterozygous, compoundheterozygous or homozygous mutations.

In one embodiment the GBA-associated disorder is associated with theL444P GBA gene mutation (L444P/, L444P/+ or L444P/L444P). A heterozygousGBA allele containing the L444P mutation may be referred to as L444P/+.

In one embodiment the GBA-associated disorder is associated with theD409H GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theD409V GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theE235A GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theE340A GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theE326K GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theN370S GBA gene mutation. A homozygous GBA allele containing the N370Smutation may be referred to as N370S/N370S. A heterozygous GBA allelecontaining the N370S mutation may be referred to as N370S/+.

In one embodiment the GBA-associated disorder is associated with theN370S/1-BP ins 84G GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theV394L GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theA456P GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theV460V GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theC342G GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theG325R GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theP415R GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theY133* GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theF213I GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with theN188S and/or IVS2+1G>A/N188S GBA gene mutation.

In one embodiment the GBA-associated disorder is associated with one ormore GBA gene mutations without accompanying reduction in GBA enzymeactivity.

In one embodiment the GBA-associated disorder is associated with reducedGBA enzyme activity and said GBA gene is wild-type. In one embodimentthe GBA-associated disorder is associated with idiopathic reduced GBAenzyme activity. A wild type GBA allele may be referred to as (+/+) (noGBA mutation).

In one embodiment the GBA-associated disorder is associated with reducedGBA activity due to suppression of activity of the protein.

In one embodiment the GBA-associated disorder is associated with reducedGBA activity due to repression of transcription or translation of thegene/protein.

In one embodiment the GBA-associated disorder is associated with reducedGBA activity and said GBA gene is wild-type, and the reduction in GBAactivity is due to suppression of activity of the protein and/orrepression of transcription or translation of the gene/protein.

In one embodiment the GBA-associated disorder is an individual with aheterozygous GBA allele containing one or more mutations selected fromthe group consisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S,N370S/1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*,F213I, N188S and IVS2+1G>A/N188S.

In one embodiment the GBA-associated disorder is an individual with aheterozygous GBA allele containing the L444P, A456P, V460V mutations incis.

In one embodiment the GBA-associated disorder is a L444P, A456P, V460VHeterozygote.

In one embodiment the GBA-associated disorder is heterozygous for thecomplex GBA allele L444P, A456P, V460V.

In one embodiment the GBA-associated disorder is a GBA mutation carrier.In one embodiment the GBA-associated disorder is an obligate carrier. Inone embodiment the GBA mutation carrier is clinically unaffected re.Gaucher's disease.

In one embodiment the GBA-associated disorder is a clinically unaffectedgrand-parent, parent, sibling or child of a Gaucher's disease patient.

In one embodiment the GBA-associated disorder is a clinically unaffectedparent or sibling of a Gaucher's disease patient.

In one embodiment the GBA-associated disorder is an individual with ahomozygous or compound heterozygous GBA allele containing one or moremutations selected from the group consisting of L444P, D409H, D409V,E235A, E340A, E326K, N370S, N370S/1-BP ins 84G, V394L, A456P, V460V,C342G, G325R, P415R, Y133*, F213I, N188S and IVS2+1G>A/N188S, whereinsaid individual remain clinically unaffected re Gaucher's disease.

In one embodiment the GBA-associated disorder is an individual with ahomozygous GBA allele containing the N370S/N370S mutation.

In one embodiment there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of treating a glucocerebrosidase (GBA)-associated disorder,such as a glucocerebrosidase (GBA)-associated disorder other thanGaucher's disease (GD).

In one embodiment the glucocerebrosidase (GBA)-associated disorder isGBA-associated parkinsonism.

In one embodiment the GBA-associated disorder is a GBA-associated Lewybody disorder, such as a GBA-associated Lewy body disorder selected fromthe group consisting of GBA-associated Parkinson's disease,GBA-associated dementia with Lewy bodies, and GBA-associated multiplesystem atrophy.

In one embodiment there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of treating a GBA-associated alpha-synucleinopathy.

A GBA-associated alpha-synucleinopathy may be defined herein as analpha-synucleinopathy having an association with the level and/oractivity of GBA enzyme. In one embodiment the alpha-synucleinopathypresents with reduced GBA levels and/or activity, which is associatedwith an increase in alpha-synuclein. In one embodiment the treatmentwith arimoclomol reduces alpha-synuclein aggregation. In one embodimentthe treatment with arimoclomol increases GBA activity and/or levels.

In one embodiment there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of treating a GBA-associated alpha-synucleinopathy selectedfrom the group consisting of GBA-associated Parkinson's disease (PD),GBA-associated dementia with Lewi bodies (DLB) and GBA-associatedmultiple system atrophy (MSA).

In one embodiment there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of treating Parkinson's disease, in particularGBA-associated Parkinson's disease.

In one embodiment the GBA-associated Parkinson's disease is Parkinson'sdisease associated with reduced GBA enzyme levels and/or activity.

In one embodiment the GBA-associated Parkinson's disease is Parkinson'sdisease associated with one or more GBA gene mutations. In oneembodiment the individual with GBA-associated Parkinson's disease remainclinically unaffected re Gaucher's disease.

In one embodiment the GBA-associated Parkinson's disease is Parkinson'sdisease associated with a heterozygous GBA gene mutation. In oneembodiment the GBA-associated disorder is an individual having one ormore heterozygous GBA gene mutations who remain clinically unaffected reGaucher's disease.

In one embodiment the GBA-associated Parkinson's disease is Parkinson'sdisease associated with a homozygous GBA gene mutation. In oneembodiment the GBA-associated disorder is an individual having one ormore homozygous and/or compound heterozygous GBA gene mutations whoremain clinically unaffected re Gaucher's disease.

In one embodiment the GBA-associated disorder is a genetically high-riskParkinson's disease GBA genotype. In one embodiment the GBA-associateddisorder is GBA-deficient Parkinson's disease (PD-GBA). In oneembodiment the GBA-associated disorder is Parkinson's disease patientswith heterozygous GBA alleles.). In one embodiment the GBA-associateddisorder is Parkinson's disease patients with homozygous GBA alleles,clinically unaffected re Gaucher's disease.

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a GBA gene mutation selected fromthe group consisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S,V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S andIVS2+1G>A/N188S. Said one or more mutations in the GBA gene can beheterozygous, compound heterozygous or homozygous mutations. In oneembodiment the individual presenting with the GBA gene mutation isclinically unaffected re Gaucher's disease.

In one embodiment the individual having GBA-associated associatedParkinson's disease has a GBA gene mutation selected from the groupconsisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S, V394L,A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S andIVS2+1G>A/N188S.

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a N370S GBA gene mutation.

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a heterozygous N370S GBA genemutation (N370S/+).

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a homozygous N370S GBA gene mutation(N370S/N370S).

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a heterozygous L444P GBA genemutation.

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a heterozygous A456P GBA genemutation.

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a heterozygous V460V GBA genemutation.

In one embodiment the GBA-associated associated Parkinson's disease isParkinson's disease associated with a heterozygous E326K GBA genemutation.

In one embodiment the GBA-associated disorder is Parkinson's diseaseassociated with idiopathic reduced GBA activity and/or levels. In oneembodiment the GBA-associated disorder is Parkinson's disease withidiopathic reduced GBA activity and/or levels, wherein no GBA genemutations are identified.

Also provided herewith is an active pharmaceutical ingredient selectedfromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of one or more of

-   -   increasing GBA activity,    -   increasing GBA levels (or amount),    -   increasing the amount of active mutant GBA,    -   increasing the amount of active wild type GBA,    -   enhancing folding of ER-retained mutant GBA,    -   increasing the amount of processed/maturated GBA,    -   increasing the amount of matured (post-ER) GBA, and/or    -   increasing the amount of matured GBA reaching the lysosomes.

In one embodiment arimoclomol is for use in a method of increasing GBAlevels and/or activity in an individual having a GBA-associateddisorder, such as a GBA-associated alpha-synucleinopathy, such asGBA-associated Parkinson's disease.

In one embodiment said GBA activity is increased to 50% or more ofhypothetical wild-type activity levels, such as 50-60%, such as 60-70%,such as 70-80%, such as 80-90%, such as 90-100%, such as 100-110%, suchas 110-120%, such as 120-130%, such as 130-140%, such as 140-150% ofhypothetical wild-type activity levels.

In one embodiment said GBA activity is increased to hypotheticalwild-type activity levels or more.

In one embodiment said GBA activity is increased at least 10%, such asat least 20%, for example at least 30%, such as at least 40%, forexample at least 50%, such as at least 60%, for example at least 70%,such as at least 80%, for example at least 90%, such as at least 100%,for example at least 110%, such as at least 120%, for example at least130%, such as at least 140%, for example at least 150%, such as at least160%, for example at least 170%, such as at least 180%, for example atleast 190%, such as at least 200%, for example at least 210%, such as atleast 220%, for example at least 230%, such as at least 240%, forexample at least 250%, such as at least 260%, for example at least 200%,such as at least 270%, for example at least 280%, such as at least 290%,for example at least 300%.

In one embodiment said GBA level (or amount) is increased to 50% or moreof hypothetical wild-type levels, such as 50-60%, such as 60-70%, suchas 70-80%, such as 80-90%, such as 90-100%, such as 100-110%, such as110-120%, such as 120-130%, such as 130-140%, such as 140-150% ofhypothetical wild-type levels.

In one embodiment said GBA level is increased to hypothetical wild-typelevels or more.

In one embodiment said GBA level and/or activity is increased at least1.5-fold, such as at least 2-fold, for example at least 2.5-fold, suchas at least 3-fold.

In one embodiment said GBA level (or amount) is increased at least 10%,such as at least 20%, for example at least 30%, such as at least 40%,for example at least 50%, such as at least 60%, for example at least70%, such as at least 80%, for example at least 90%, such as at least100%, for example at least 110%, such as at least 120%, for example atleast 130%, such as at least 140%, for example at least 150%, such as atleast 160%, for example at least 170%, such as at least 180%, forexample at least 190%, such as at least 200%, for example at least 210%,such as at least 220%, for example at least 230%, such as at least 240%,for example at least 250%, such as at least 260%, for example at least200%, such as at least 270%, for example at least 280%, such as at least290%, for example at least 300%.

Also provided herewith is an active pharmaceutical ingredient selectedfromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of reducing alpha-synuclein aggregation.

Preventive Use

In another aspect there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of reducing risk in an individual of developing aglucocerebrosidase (GBA)-associated disorder other than Gaucher'sdisease, wherein said individual has reduced GBA enzyme level and/oractivity.

In one embodiment said individual has GBA levels and/or activity lowerthan hypothetical wild-type levels.

In one embodiment said individual has GBA levels (or amount) lower thanhypothetical wild-type levels.

In one embodiment said individual has GBA activity lower thanhypothetical wild-type activity levels.

In one embodiment said individual has GBA levels and/or activity higherthan clinically affected levels and/or activity in a patient withGaucher's disease.

In one embodiment said individual has GBA levels and/or activity lowerthan hypothetical wild-type levels and/or activity, yet higher thanclinically affected levels and/or activity in a patient with Gaucher'sdisease.

In one embodiment said individual has reduced GBA activity to the samedegree as a GBA gene mutation carrier (heterozygous GBA mutation), suchas a clinically unaffected carrier, such as an obligate carrier.

In one embodiment said individual has reduced GBA levels to the samedegree as a GBA gene mutation carrier (heterozygous GBA mutation), suchas a clinically unaffected carrier, such as an obligate carrier.

In one embodiment said individual with reduced GBA levels and/oractivity has one or more heterozygous GBA gene mutations.

In one embodiment said individual with reduced GBA levels and/oractivity has one or more homozygous or compound heterozygous GBA genemutations.

In one embodiment said individual has reduced GBA activity and/or levelto a certain extent of hypothetical wild type levels.

In one embodiment said individual has GBA activity and/or levels ofabout 5 to 95% or 10 to 90% of hypothetical wild type levels, such as 5to 10%, such as 10 to 20%, such as 20 to 30%, such as 30 to 40%, such as40 to 50%, such as 50 to 60%, such as 60 to 70%, such as 70 to 80%, suchas 80 to 90%, such as 90 to 95% of hypothetical wild type activityand/or levels.

In one embodiment said individual has GBA activity and/or levels ofabout 25 to 75% of hypothetical wild type levels. In one embodiment saidindividual has GBA activity and/or levels of about 50% of hypotheticalwild type levels.

In one embodiment said individual has GBA activity and/or levels ofabout 10 of hypothetical wild type levels and/or activity, such as 20%,such as 30%, such as 40%, such as 50%, such as 60%, such as 70%, such as80%, such as 90% of hypothetical wild type levels and/or activity.

In one embodiment said GBA-associated disorder is a GBA-associatedalpha-synucleinopathy, such as a GBA-associated alpha-synucleinopathyselected from the group consisting of GBA-associated Parkinson'sdisorder (PD), GBA-associated dementia with Lewi bodies (DLB) andGBA-associated multiple system atrophy (MSA).

In one embodiment there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of reducing risk in an individual of developing Parkinson'sdisease, in particular GBA-associated Parkinson's disease, wherein saidindividual has reduced GBA enzyme levels and/or activity.

In one embodiment said individual has one or more heterozygous GBA genemutations. In one embodiment said individual has one or moreheterozygous GBA gene mutations selected from the group consisting ofL444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S/1-BP ins 84G,V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S andIVS2+1G>A/N188S. In one embodiment said individual has a heterozygousL444P GBA gene mutation. In one embodiment said individual has aheterozygous E326K GBA gene mutation. In one embodiment said individualhas a heterozygous N370S GBA gene mutation.

In one embodiment said individual has one or more homozygous GBA genemutations. In one embodiment said individual has one or more homozygousGBA gene mutations selected from the group consisting of L444P, D409H,D409V, E235A, E340A, E326K, N370S, N370S/1-BP ins 84G, V394L, A456P,V460V, C342G, G325R, P415R, Y133*, F213I, N188S and IVS2+1G>A/N188S. Inone embodiment said individual has a homozygous N370S GBA gene mutation.

In one embodiment there is provided an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of reducing risk in an individual of developingGBA-associated Parkinson's disease, wherein said individual is a patientwith Gaucher's disease, such as Gaucher's disease type I, type II ortype III.

GBA Activity

Glucocerebrosidase activity can be assessed by methods known in the art.For example, the glucocerebrosidase activity may be measured from thecerebral spinal fluid of mammals. In some embodiments, the mammal iswild-type for the GBA gene. The term “wild-type” refers to a gene orprotein with no detectable mutations known to affect the level and/orenzymatic activity of the protein.

When the gene is found to be wild-type, but a reduction inglucocerebrosidase activity is observed, the reduction in activity maybe due to suppression of activity of the protein or repression oftranscription or translation of the gene/protein. These mechanisms arewell known in the art. For example, the production of the protein may berepressed by aberrant cellular mechanism. Alternatively, the protein maybe modified in the cell which causes reduced or loss of enzymaticactivity.

Arimoclomol

Reference to arimoclomol herein encompasses an active pharmaceuticalingredient (API) selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride (arimoclomol), its stereoisomers and the acid addition saltsthereof. Arimoclomol is further described in e.g. WO 00/50403.

Arimoclomol refers to the base compoundN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its optically active (+) or (−) enantiomer, a mixture of theenantiomers of any ratio, and the racemic compound, furthermore, theacid addition salts formed from any of the above compounds with mineralor organic acids constitute objects of the present invention. Allpossible geometrical isomer forms ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride belong to the scope of the invention. The term “thestereoisomers ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride” refers to all possible optical and geometrical isomers of thecompound.

If desired, theN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride or one of its optically active enantiomers can be transformedinto an acid addition salt with a mineral or organic acid, by knownmethods.

In one embodiment the active pharmaceutical ingredient is the racemateofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.

In one embodiment the active pharmaceutical ingredient is an opticallyactive stereoisomer ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.

In one embodiment the active pharmaceutical ingredient is an enantiomerofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.

In one embodiment the active pharmaceutical ingredient is selected fromthe group consisting of(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride and(−)-(S)—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.

In one embodiment the active pharmaceutical ingredient is an acidaddition salt ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.

In one embodiment the active pharmaceutical ingredient is selected fromthe group consisting ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate (also known as BRX-345), andN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate (also known as BRX-220).

In one embodiment the active pharmaceutical ingredient is selected fromthe group consisting of(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate;(−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate;(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate; and(−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate.

Composition

Whilst it is possible for the active pharmaceutical ingredient to beadministered as the raw chemical, it is in some embodiments preferred topresent them in the form of a pharmaceutical formulation. Accordingly,also provided herewith is a composition, such as a pharmaceuticalcomposition, i.e. a pharmaceutically safe composition, comprising anactive pharmaceutical ingredient as defined herein. The composition inone embodiment comprises a pharmaceutically and/or physiologicallyacceptable carriers or excipients.

Pharmaceutical compositions containing a bioactive agent of the presentinvention may be prepared by conventional techniques, e.g. as describedin Remington: The Science and Practice of Pharmacy, 20^(th) Edition,Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2000.

It is thus an aspect to provide a composition, such as a pharmaceuticalcomposition, comprising an active pharmaceutical ingredient selectedfromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof(arimoclomol), for use in the treatment of a glucocerebrosidase(GBA)-associated disorder other than Gaucher's disease (GD) as definedherein.

Administration and Dosage

An active pharmaceutical ingredient or composition comprising the sameas defined herein is in one embodiment administered to individuals inneed thereof in pharmaceutically effective doses or a therapeuticallyeffective amount.

A therapeutically effective amount of an active pharmaceuticalingredient is in one embodiment an amount sufficient to cure, prevent,reduce the risk of, alleviate or partially arrest the clinicalmanifestations of a given disease or disorder and its complications. Theamount that is effective for a particular therapeutic purpose willdepend on the severity and the sort of the disorder as well as on theweight and general state of the subject. An amount adequate toaccomplish this is defined as a “therapeutically effective amount”.

In one embodiment, the composition is administered in doses of 1 μg/dayto 100 mg/day; such as 1 μg/day to 10 μg/day, such as 10 μg/day to 100μg/day, such as 100 μg/day to 250 μg/day, such as 250 μg/day to 500μg/day, such as 500 μg/day to 750 μg/day, such as 750 μg/day to 1mg/day, such as 1 mg/day to 2 mg/day, such as 2 mg/day to 5 mg/day, orsuch as 5 mg/day to 10 mg/day, such as 10 mg/day to 20 mg/day, such as20 mg/day to 30 mg/day, such as 30 mg/day to 40 mg/day, such as 40mg/day to 50 mg/day, such as 50 mg/day to 75 mg/day, such as 75 mg/dayto 100 mg/day, such as 100 mg/day to 150 mg/day, such as 150 mg/day to200 mg/day, or such as 200 mg/day to 250 mg/day, such as 250 mg/day to300 mg/day, such as 300 mg/day to 400 mg/day, such as 400 mg/day to 500mg/day, such as 500 mg/day to 600 mg/day, such as 600 mg/day to 700mg/day, such as 700 mg/day to 800 mg/day, such as 800 mg/day to 900mg/day, such as 900 mg/day to 1000 mg/day.

In one embodiment, the active pharmaceutical ingredient or compositionis administered at a dose of 1 μg/kg body weight to 100 mg/kg bodyweight; such as 1 to 10 μg/kg body weight, such as 10 to 100 μg/day,such as 100 to 250 μg/kg body weight, such as 250 to 500 μg/kg bodyweight, such as 500 to 750 μg/kg body weight, such as 750 μg/kg bodyweight to 1 mg/kg body weight, such as 1 mg/kg body weight to 2 mg/kgbody weight, such as 2 to 5 mg/kg body weight, such as 5 to 10 mg/kgbody weight, such as 10 to 20 mg/kg body weight, such as 20 to 30 mg/kgbody weight, such as 30 to 40 mg/kg body weight, such as 40 to 50 mg/kgbody weight, such as 50 to 75 mg/kg body weight, or such as 75 to 100mg/kg body weight.

In one embodiment, a dose is administered one or several times per day,such as from 1 to 6 times per day, such as from 1 to 5 times per day,such as from 1 to 4 times per day, such as from 1 to 3 times per day,such as from 1 to 2 times per day, such as from 2 to 4 times per day,such as from 2 to 3 times per day. In one embodiment, a dose isadministered less than once a day, such as once every second day or oncea week.

Routes of Administration

It will be appreciated that the preferred route of administration willdepend on the general condition and age of the subject to be treated,the nature of the condition to be treated, the location of the tissue tobe treated in the body and the active ingredient chosen.

Systemic Treatment

In one embodiment, the route of administration allows for introducingthe bioactive agent into the blood stream to ultimately target the sitesof desired action.

In one embodiment the routes of administration is any suitable route,such as an enteral route (including the oral, rectal, nasal, pulmonary,buccal, sublingual, transdermal, intracisternal and intraperitonealadministration), and/or a parenteral route (including subcutaneous,intramuscular, intrathecal, intravenous and intradermal administration).

Appropriate dosage forms for such administration may be prepared byconventional techniques.

Parenteral Administration

Parenteral administration is any administration route not being theoral/enteral route whereby the bioactive agent avoids first-passdegradation in the liver. Accordingly, parenteral administrationincludes any injections and infusions, for example bolus injection orcontinuous infusion, such as intravenous administration, intramuscularadministration or subcutaneous administration. Furthermore, parenteraladministration includes inhalations and topical administration.

Accordingly, the active pharmaceutical ingredient or composition is inone embodiment administered topically to cross any mucosal membrane ofan animal, e.g. in the nose, vagina, eye, mouth, genital tract, lungs,gastrointestinal tract, or rectum, for example the mucosa of the nose,or mouth, and accordingly, parenteral administration may also includebuccal, sublingual, nasal, rectal, vaginal and intraperitonealadministration as well as pulmonal and bronchial administration byinhalation or installation. In some embodiments, the bioactive agent isadministered topically to cross the skin.

In one embodiment, the intravenous, subcutaneous and intramuscular formsof parenteral administration are employed.

Local Treatment

In one embodiment, the active pharmaceutical ingredient t or compositionis used as a local treatment, i.e. is introduced directly to the site(s)of action. Accordingly, the active pharmaceutical ingredient may beapplied to the skin or mucosa directly, or may be injected into the siteof action, for example into the diseased tissue or to an end arteryleading directly to the diseased tissue.

Combination Treatment

It is also an aspect to provide an active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof(arimoclomol), for use in a method of treating a glucocerebrosidase(GBA)-associated disorder other than Gaucher's disease (GD), incombination with other treatment modalities.

Thus, in one embodiment, the active pharmaceutical ingredient isadministered to an individual in need thereof in combination with atleast one other treatment modality, such as conventional or knowntreatment modalities for (GBA)-associated disorders includingGBA-associated alpha-synucleinopathies such as GBA-associatedParkinson's disease (PD), GBA-associated dementia with Lewi bodies (DLB)and GBA-associated multiple system atrophy (MSA).

Administering more than one treatment modality in combination may occureither simultaneously, or sequentially. Simultaneous administration maybe two compounds comprised in the same composition or comprised inseparate compositions, or may be one composition and one other treatmentmodality performed essentially at the same time. Sequentialadministration means that the more than one treatment modalities areadministered at different time points, such as administering onetreatment modality first, and administering the second treatmentmodality subsequently. The time frame for administering more than onetreatment modality sequentially may be determined by a skilled person inthe art for achieving the optimal effect, and may in one embodiment bebetween 30 minutes to 72 hours.

The treatment modalities in the form of chemical compounds may beadministered together or separately, each at its most effective dosage.Administering more than one compound may have a synergistic effect, thuseffectively reducing the required dosage of each drug.

It is also an aspect to provide a composition comprising, separately ortogether, i) an active pharmaceutical ingredient selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof(arimoclomol), and ii) other treatment modalities, for use in thetreatment of (GBA)-associated disorders including GBA-associatedalpha-synucleinopathies such as GBA-associated Parkinson's disease (PD),GBA-associated dementia with Lewi bodies (DLB) and GBA-associatedmultiple system atrophy (MSA).

In one embodiment other treatment modalities, or conventional or knowntreatment modalities, are referred to as further active ingredients.

In one embodiment the active pharmaceutical ingredient selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof(arimoclomol), is administered in combination with, and/or formulated asa combination product, with one or more further active ingredients.

In one embodiment the further active ingredient is selected from one ormore active ingredients known and/or employed in the treatment of(GBA)-associated disorders including GBA-associatedalpha-synucleinopathies such as GBA-associated Parkinson's disease (PD),GBA-associated dementia with Lewi bodies (DLB) and GBA-associatedmultiple system atrophy (MSA).

In one embodiment the further active ingredient is a compound used forthe treatment of Parkinson's disease. In one embodiment said compoundused for the treatment of Parkinson's disease is selected from the groupconsisting of dopamine, L-DOPA, levodopa, dopamine receptor agonists,carboxylase inhibitors such as carbidopa or benserazide, NMDAantagonists such as for example amatidine (Symmetrel), catechol-O-methyltransferase (COMT) inhibitors such as for example tolcapone andentacapone, MAO-B inhibitors such as for example selegiline andrasagiline, Carbidopa-levodopa, Anticholinergics and Amantadine.

In one embodiment the further active ingredient is a compound used forthe treatment of Gaucher's disease. In one embodiment the further activeingredient is selected from the group consisting of enzyme replacementtherapies, allosteric chaperones, pharmacological chaperones andsubstrate reduction therapies. In one embodiment said further activeingredient is selected from the group consisting of miglustat (Zavesca),imiglucerase (Cerezyme), eliglustat (Cerdelga), VPRIV, taliglucerasealfa (Elelyso) and velaglurase alpha.

EXAMPLES Example 1: Dose-Dependent Response in Gaucher Type IIHeterozygotes (Parkinsons Disease Genotype)—BiP and GBA InductionMaterials and Methods Cell Culture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

GD Cell line GBA mutation Age at Sampling type GM00877 L444P/L444P,A456P, V460V 1Y II GM00878 L444P, A456P, V460V — carrier

Western Blotting

Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4°C. Cell pellets were lysed in 1× Extraction buffer (Enzo Life Science)containing protease inhibitors, sonicated and cleared by centrifugationat 13000 rpm for 10 min at 4° C. Protein concentration measured by theBCA assay. Samples containing approx. 10-20 μg protein were diluted inglycoprotein denaturing buffer (New England Biolabs) and denatured byincubation for 10 min at 100° C. Samples were incubated with or withoutEndoH (New England Biolabs) for 1 h at 37° C., Laemmli sample buffer wasadded and the samples were subjected to SDS-PAGE using the TGX gelsystem (Bio-Rad). After transfer to a nitrocellulose membrane(Trans-Blot Turbo, Bio-Rad), the membranes were stained briefly withPonceau S, and subsequently blocked in 5% skim-milk in PBS+0.1% tween(PBS-T). Incubation with primary antibodies (1:500 to 1:2000 dilution)was performed on parafilm-coated glass plates overnight at 4° C. Afterwashing in PBS-T the membranes were incubated 1 h with secondaryantibody diluted 1:10,000 in 5% skim milk in PBS-T. The blots weredeveloped using SuperSignal™ West Dura Extended Duration Substrate (Lifetechnologies) and visualized using a G-box system (Syngene).

Results Arimoclomol-Induced Dose-Dependent Increase in ER Hsp70 (BiP) inPrimary Cells

Arimoclomol is reported to increase the expression levels of heat-shockproteins, e.g. heat-shock protein 70 (HSP70) (Kieran et al., NatureMedicine, 2004).

To assess the effect of arimoclomol on the ER Hsp70 (BiP) expressionlevel in primary cells, human fibroblasts from individual with aheterozygous GBA allele containing the L444P, A456P, V460V mutations incis (carrier, clinically unaffected re. Gaucher's disease) were treatedwith 0, 10, 50 or 200 μM arimoclomol for 14 days. Cells were thenharvested for western blot analysis. A lysate from untreated normalhuman fibroblasts was used for control.

Our results demonstrate that arimoclomol dose-dependently increases BiPexpression levels in a human fibroblasts cell line heterozygous for thecomplex GBA allele L444P, A456P, V460V. This suggests that arimoclomolvia BiP-upregulation can lead to an enhanced folding of ER-retainedmutant GBA.

Arimoclomol-Induced Dose-Dependent Increase in GBA Enzyme Amount inPrimary Cells

The effect of arimoclomol on GBA protein levels was also evaluated inthe human fibroblasts cell line heterozygous for the complex GBA alleleL444P, A456P, V460V. In line with an upregulation of the ER chaperoneBiP, a dose-dependent increase in the total level of GBA is seen inarimoclomol-treated cells.

Example 2: Dose-Dependent Response on GBA Activity in Gaucher Type IIHomozygotes and Heterozygotes (GTII and High-Risk Parkinsons DiseaseGenotype) Materials and Methods Cell Culture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

GBA Activity Assay

GBA activity was measured using the “intact cell” GBA assay using the4-Methylumbelliferyl beta-D-glucopyranoside (4-MUG) substrate (Mu et al,Cell, 2008). Briefly, fibroblasts were seeded in 12 well plates andtreated in biological triplicate with indicated concentrations ofarimoclomol for 4 weeks. Medium was replenished with fresh compoundevery 2-3 days and the cells were split twice during the experiment.After 4 weeks of treatment, cells were transferred to 96 well plates andGBA activity was measured using 4-MUG as substrate at pH 4.0. Thereleased 4-MU fluorophore was quantified as Fluorescence Units (FLU) andnormalized to cell density using crystal violet staining of a parallelplate. The normalized data is reported as Arbitrary Units (mean±SD).

Results

The effect of arimoclomol on GBA activity in primary cells withmutations of GBA was evaluated in fibroblasts from a Type II Gaucherpatient with the genotype L444P/L444P, A456P, V460V. We observed thatarimoclomol treatment increased GBA activity in a dose-dependent manner.Notably, the increase in GBA activity induced by 50 μM arimoclomolcorresponds to the activity level of cells heterozygous for the L444P,A456P, V460V allele (marked by grey line in FIG. 3) from anon-symptomatic individual.

Importantly, arimoclomol also increases the GBA activity in primaryfibroblasts that are heterozygous for the L444P, A456P, V460V allele.This result demonstrates that GBA activity can be increased even incells from mutant GBA heterozygotes (carriers).

Example 3: Dose-Dependent Response on GBA Activity in Gaucher Type I,Type II and Type II Homozygotes Materials and Methods Cell Culture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling Disease GM08760 L444P,E326K/L444P, E326K  1Y GD TII GM10915 L444P/L444P  7Y GD TIII GM01607N370S/V394L 30Y GD TI GM00372 N370S/1-BP ins 84G 29Y GD TI GM02627G325R/C342G  3Y GD TII GM01260 L444P/P415R 11M GD TII

GBA Activity Assay

GBA activity was measured using the “intact cell” GBA assay using the4-Methylumbelliferyl beta-D-glucopyranoside (4-MUG) substrate (Mu et al,Cell, 2008). Briefly, fibroblasts were seeded in 96 well plates andtreated in biological triplicate with indicated concentrations ofarimoclomol for 5 days. Medium was replenished with fresh compound every2-3 days. GBA activity was measured using 4-MUG as substrate at pH 4.0.The released 4-MU fluorophore was quantified as Fluorescence Units (FLU)and normalized to cell density using crystal violet staining of aparallel plate. The normalized data is reported as fold change relativeto mock-treated control cells (mean±SD).

Results

The effect of arimoclomol on GBA activity in primary cells withadditional mutations of GBA was evaluated by treating cells of theindicated genotype with arimoclomol. Our data show that arimoclomoldose-dependently increases GBA activity in two GD type I cell lines:N370S/V394L and N370S/1-BP ins 84G. Importantly, since the 1-BP ins 84Gis considered to be a null-allele, these results demonstrate thatarimoclomol increases the activity of the N370S mutation.

A dose-dependent effect of arimoclomol is also seen for GBA activity inprimary cells from GD type II/III patients which are either homozygotesfor L444P (L444P/L444P) or compound heterozygotes for the GBA mutationsG325R/C342G or P415R/L444P. A less pronounced increase of GBA activityis found in arimoclomol-treated type II GD cells homozygous for theE326K, L444P allele.

Example 4: Dose-Dependent Response on GBA Activity in Primary Cells froma Parkinson Disease Patient with a Heterozygous GBA Allele Containingthe N370S Mutation Materials and Methods Cell Culture

The primary human fibroblast cell line was cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. The cellswere passaged 1 time/week with a split ratio of 1:2. Cells were used forexperiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling Disease ND34263 N370S 65Y PD

GBA Activity Assay

GBA activity was measured using the “intact cell” GBA assay using the4-Methylumbelliferyl beta-D-glucopyranoside (4-MUG) substrate (Mu et al,Cell, 2008). Briefly, fibroblasts were seeded in 96 well plates andtreated in biological triplicate with indicated concentrations ofarimoclomol for 5 days. Medium was replenished with fresh compound every2-3 days. GBA activity was measured using 4-MUG as substrate at pH 4.0.The released 4-MU fluorophore was quantified as Fluorescence Units (FLU)and normalized to cell density using crystal violet staining of aparallel plate. The normalized data is reported as fold change relativeto mock-treated control cells (mean±SD).

Results

To investigate the effect of arimoclomol on the N370S mutation inParkinson disease, primary cells from a PD patient with N370S mutationwas treated with arimoclomol. Our data show that arimoclomoldose-dependently increases N370S GBA activity in cells from a PDpatient.

Example 5: Dose-Dependent Response on GBA Activity in Primary Cells fromHealthy Individuals without GBA Mutations (+/+) Materials and MethodsCell Culture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling Disease GM00498 — 3Y — GM05659 —1Y — GM08401 — 75Y  —

GBA Activity Assay

GBA activity was measured using the “intact cell” GBA assay using the4-Methylumbelliferyl beta-D-glucopyranoside (4-MUG) substrate (Mu et al,Cell, 2008). Briefly, fibroblasts were seeded in 96 well plates andtreated in biological triplicate with indicated concentrations ofarimoclomol for 5 days. Medium was replenished with fresh compound every2-3 days. GBA activity was measured using 4-MUG as substrate at pH 4.0.The released 4-MU fluorophore was quantified as Fluorescence Units (FLU)and normalized to cell density using crystal violet staining of aparallel plate. The normalized data is reported as fold change relativeto mock-treated control cells (mean±SD).

Results

To investigate the effect of arimoclomol on WT GBA protein, primarycells from healthy individuals without GBA mutations (+/+) were treatedwith arimoclomol. We observed that arimoclomol treatment increased WTGBA activity in a dose-dependent manner in all three cell lines albeitto different magnitude.

Example 6: Arimoclomol-Induced Increase in Activity-Based Probe Labelingof Active GBA in Gaucher Disease TI/TII/TIII Materials and Methods CellCulture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO2) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling Disease GM01607 N370S/V394L 30Y GD TI GM02627 G325R/C342G 3Y GD TII GM10915 L444P/L444P 7Y GD TIIIGBA Labeling with ME569

Active GBA can be selectively labeled with the fluorescentactivity-based probe (ABP) ME569 (Witte et al, 2010). Briefly,fibroblasts were seeded in dishes and treated in biological duplicateswith the indicated concentrations of arimoclomol for 5 days. Medium wasreplenished with fresh compound every 2-3 days. Cells were collected inPBS, proteins were extracted and the concentrations were determinedusing the BCA assay. Equal amount of total protein was incubated withME569 for 30 minutes at 37° C. Loading buffer was added, samples wereincubated for 5 min at 98° C. and then subjected to SDS-PAGE using theTGX gel system (Bio-Rad). After gel electrophoresis, fluorescence wasdetected using red LEDs/705M filter (G-box, Syngene). The amount oflabeled GBA was quantified using the software GeneTools v.4.03.01.0 fromSyngene. The normalized data is reported as fold change relative tomock-treated control cells (mean±SEM, n=3-4).

Results

The effect of arimoclomol on the amount of GBA labeled with fluorescentABP was evaluated in primary cells from GD patients of the indicatedgenotype. Our data show that arimoclomol dose-dependently increases GBAlabeling in the GD TI cell line (N370S/V394L) and the GD TII cell line(G325R/C342G). Only the high dose of arimoclomol was evaluated in the GDTII cell line (homozygotes for L444P) and also in this cell line,arimoclomol increases the amount of GBA that can be labeled withfluorescent ABP.

Taken together, these data show that arimoclomol increases the amount ofactive mutant GBA in primary cells from all three types of GaucherDisease (TI type 1, TII type II and TIII type III).

Example 7: Dose-Dependent Response in Gaucher Type I—BiP and GBAInduction Materials and Methods Cell Culture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling GD type GM00372 N370S/1-BP ins84G 29Y I GM01607 N370S/V394L 30Y I

Western Blotting

Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4°C. Cell pellets were lysed in lysis buffer (Enzo Life Science)containing protease inhibitors, sonicated and cleared by centrifugationat 13000 rpm for 10 min at 4° C. Protein concentration was measured bythe BCA assay. Samples containing approx. 10-20 μg protein were dilutedin glycoprotein denaturing buffer (New England Biolabs) and denatured byincubation for 10 min at 100° C. Samples were incubated with or withoutEndoH (New England Biolabs) for 1 h at 37° C., Laemmli sample buffer wasadded and the samples were subjected to SDS-PAGE using the TGX gelsystem (Bio-Rad). After transfer to a nitrocellulose membrane(Trans-Blot Turbo, Bio-Rad), the membranes were stained briefly withPonceau S, and subsequently blocked in 5% skim-milk in PBS+0.1% tween(PBS-T). Incubation with primary antibodies (1:500 to 1:2000 dilution)was performed on parafilm-coated glass plates overnight at 4° C. Afterwashing in PBS-T the membranes were incubated 1 h with secondaryantibody diluted 1:10,000 in 5% skim milk in PBS-T. The blots weredeveloped using SuperSignal™ West Dura Extended Duration Substrate (Lifetechnologies) and visualized using a G-box system (Syngene).

Results Arimoclomol-Induced Dose-Dependent Increase in ER Hsp70 (BiP) inType I GD Primary Cells

Arimoclomol is reported to increase the expression levels of heat-shockproteins, e.g. heat-shock protein 70 (HSP70) (Kieran et al., NatureMedicine, 2004). To assess the effect of arimoclomol on the ER Hsp70(BiP) expression level in primary cells, human fibroblasts from GD TIpatients were treated with 0, 25, 100, 200 or 400 μM arimoclomol(N370S/V394L) or 0, 100, 200 or 400 μM arimoclomol (N370S/1-BP ins 84G)for 5 days. Cells were then harvested for western blot analysis.

Our results demonstrate that arimoclomol dose-dependently increases BiPexpression levels in human fibroblasts cell lines from type I Gaucherindividuals. This suggests that arimoclomol via BiP-upregulation canlead to an enhanced folding of ER-retained mutant GBA.

Arimoclomol-Induced Dose-Dependent Increase in GBA Enzyme Amount in TypeI GD Primary Cells

The effect of arimoclomol on GBA protein levels was also evaluated inthe human fibroblasts cell line from a type I Gaucher patient with theN370S/1-BP ins 84G genotype. In line with an upregulation of the ERchaperone BiP, a dose-dependent increase in the total level of GBA isseen in arimoclomol-treated cells from this individual. Moreover, anincrease in the EndoH-resistant fraction shows that arimoclomolincreases the amount of processed/maturated GBA in the cell.

Example 8: Dose-Dependent Response in Gaucher Type II—BiP and GBAInduction Materials and Methods Cell Culture

Primary human fibroblast cell lines were cultured under standard cellculture conditions (37° C. and 5% CO₂) in DMEM supplemented withnon-essential amino acids (NEAA), 1% Pen-Strep and 12% FCS. They werepassaged 1-2 times/week with a split ratio of 1:2 or 1:3. Cells wereused for experiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling GD type GM01260 L444P/P415R 11MII GM02627 G325R/C342G  3Y II

Western Blotting

Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4°C. Cell pellets were lysed in lysis buffer (Enzo Life Science)containing protease inhibitors, sonicated and cleared by centrifugationat 13000 rpm for 10 min at 4° C. Protein concentration was measured bythe BCA assay. Samples containing approx. 10-20 μg protein were dilutedin glycoprotein denaturing buffer (New England Biolabs) and denatured byincubation for 10 min at 100° C. Samples were incubated with or withoutEndoH (New England Biolabs) for 1 h at 37° C., Laemmli sample buffer wasadded and the samples were subjected to SDS-PAGE using the TGX gelsystem (Bio-Rad). After transfer to a nitrocellulose membrane(Trans-Blot Turbo, Bio-Rad), the membranes were stained briefly withPonceau S, and subsequently blocked in 5% skim-milk in PBS+0.1% tween(PBS-T). Incubation with primary antibodies (1:500 to 1:2000 dilution)was performed on parafilm-coated glass plates overnight at 4° C. Afterwashing in PBS-T the membranes were incubated 1 h with secondaryantibody diluted 1:10,000 in 5% skim milk in PBS-T. The blots weredeveloped using SuperSignal™ West Dura Extended Duration Substrate (Lifetechnologies) and visualized using a G-box system (Syngene).

Results Arimoclomol-Induced Dose-Dependent Increase in ER Hsp70 (BiP) inType II GD Primary Cells

To assess the effect of arimoclomol on the ER Hsp70 (BiP) expressionlevel in primary cells, human fibroblasts from type II Gaucher diseasepatients were treated with the indicated concentrations of arimoclomolfor 5 days. Cells were then harvested for western blot analysis.

Our results demonstrate that arimoclomol dose-dependently increases BiPexpression levels in human fibroblasts cell lines from GD TIIindividuals with L444P/P415R or G325R/C342G genotypes. This suggeststhat arimoclomol via BiP-upregulation can lead to an enhanced folding ofER-retained mutant GBA in GD TII cells.

Arimoclomol-Induced Dose-Dependent Increase in GBA Enzyme Amount in GDTII Primary Cells

The effect of arimoclomol on GBA protein levels and maturation was alsoevaluated in GD TII primary cell lines. In line with an upregulation ofthe ER chaperone BiP, a dose-dependent increase in the total level ofGBA is seen in arimoclomol-treated cells from these individuals.Moreover, an increase in the EndoH-resistant fraction shows thatarimoclomol increases the amount of matured (post-ER) GBA in GD type IIcells.

Example 9: Dose-Dependent Response in Gaucher Type III—BiP and GBAInduction Materials and Methods Cell Culture

Primary human fibroblast cells were cultured under standard cell cultureconditions (37° C. and 5% CO₂) in DMEM supplemented with non-essentialamino acids (NEAA), 1

Pen-Strep and 12% FCS. They were passaged 1-2 times/week with a splitratio of 1:2 or 1:3. Cells were used for experiments around passage16-26 where no signs of replicative senescence were observed (visualinspection).

Cell line GBA mutation Age at Sampling GD type GM10915 L444P/L444P 7YIII

Western Blotting

Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4°C. Cell pellets were lysed in lysis buffer (Enzo Life Science)containing protease inhibitors, sonicated and cleared by centrifugationat 13000 rpm for 10 min at 4° C. Protein concentration was measured bythe BCA assay. Samples containing approx. 10-20 μg protein were dilutedin glycoprotein denaturing buffer (New England Biolabs) and denatured byincubation for 10 min at 100° C. Samples were incubated with or withoutEndoH (New England Biolabs) for 1 h at 37° C., Laemmli sample buffer wasadded and the samples were subjected to SDS-PAGE using the TGX gelsystem (Bio-Rad). After transfer to a nitrocellulose membrane(Trans-Blot Turbo, Bio-Rad), the membranes were stained briefly withPonceau S, and subsequently blocked in 5% skim-milk in PBS+0.1% tween(PBS-T). Incubation with primary antibodies (1:500 to 1:2000 dilution)was performed on parafilm-coated glass plates overnight at 4° C. Afterwashing in PBS-T the membranes were incubated 1 h with secondaryantibody diluted 1:10,000 in 5% skim milk in PBS-T. The blots weredeveloped using SuperSignal™ West Dura Extended Duration Substrate (Lifetechnologies) and visualized using a G-box system (Syngene).

Results Arimoclomol-Induced Dose-Dependent Increase in ER Hsp70 (BiP) inGD Till Primary Cells

To assess the effect of arimoclomol on the ER Hsp70 (BiP) expressionlevel in primary cells, human fibroblasts from an individual with typeIII Gaucher disease (L444P/L444P) were treated with the indicatedconcentrations of arimoclomol for 5 days. Cells were then harvested forwestern blot analysis.

Our results demonstrate that arimoclomol dose-dependently increases BiPexpression levels in this cell line homozygous for L444P. This suggeststhat arimoclomol via BiP-upregulation can lead to an enhanced folding ofER-retained mutant GBA.

Arimoclomol-Induced Dose-Dependent Increase in GBA Enzyme Amount in GDTIII Primary Cells

The effect of arimoclomol on GBA protein levels and maturation was alsoevaluated in the L444P/L444P GD TIII primary cell line. In line with anupregulation of the ER chaperone BiP, a dose-dependent increase in thetotal level of GBA is seen in arimoclomol-treated cells. Moreover, anincrease in the EndoH-resistant fraction of GBA shows that arimoclomolincreases the amount of maturated GBA in the GD TIII cells.

Taken together, these results show that GBA activity is increased byarimoclomol likely due to more mature GBA reaching the lysosomes.

Example 10: Dose-Dependent Response in BiP Expression in GBA-DeficientParkinson Disease Materials and Methods Cell Culture

Primary human fibroblast cells were cultured under standard cell cultureconditions (37° C. and 5% CO₂) in DMEM supplemented with non-essentialamino acids (NEAA), 1% Pen-Strep and 12% FCS. They were passaged 1-2times/week with a split ratio of 1:2 or 1:3. Cells were used forexperiments around passage 16-26 where no signs of replicativesenescence were observed (visual inspection).

Cell line GBA mutation Age at Sampling ND34263 N370S/N370S 65Y PD-GBA

Western Blotting

Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4°C. Cell pellets were lysed in lysis buffer (Enzo Life Science)containing protease inhibitors, sonicated and cleared by centrifugationat 13000 rpm for 10 min at 4° C. Protein concentration was measured bythe BCA assay and the samples were subjected to SDS-PAGE using the TGXgel system (Bio-Rad). After transfer to a nitrocellulose membrane(Trans-Blot Turbo, Bio-Rad), the membranes were stained briefly withPonceau S, and subsequently blocked in 5% skim-milk in PBS+0.1% tween(PBS-T). Incubation with primary antibodies (1:500 to 1:2000 dilution)was performed on parafilm-coated glass plates overnight at 4° C. Afterwashing in PBS-T the membranes were incubated 1 h with secondaryantibody diluted 1:10,000 in 5% skim milk in PBS-T. The blots weredeveloped using SuperSignal™ West Dura Extended Duration Substrate (Lifetechnologies) and visualized using a G-box system (Syngene).

Results Arimoclomol-Induced Dose-Dependent Increase in ER Hsp70 (BiP) inPD-GBA Primary Cells

To assess the effect of arimoclomol on the ER Hsp70 (BiP) expressionlevel in primary cells from an individual with GBA-deficient ParkinsonDisease (PD-GBA), human fibroblasts from an individual with PD-GBA(N370S/N370S) were treated with the indicated concentrations ofarimoclomol for 5 days. Cells were then harvested for western blotanalysis.

Our results demonstrate that arimoclomol dose-dependently increases BiPexpression levels in primary cells from an individual with PD-GBA(N370S/N370S). This suggests that arimoclomol via BiP-upregulation leadsto an enhanced folding of ER-retained mutant GBA.

Example 11: Effect of Arimoclomol on GBA Activity in PrimaryNeuronal-Like Cells from GDTI and GDTIII Individuals Materials andMethods Cell Culture

Human multipotent adult stem cells (MASCs) were isolated from GDindividuals from skin biopsies. The genotype of the MASCs is shownbelow. Cells were induced to differentiate along a neuronal fate asdescribed in Bergamin et al. Orphanet Journal of Rare Diseases 2013. Thesurface immunophenotype of stem cells was analyzed by FACS. Stem celland neuronal markers expression were evaluated by immunofluorescence.

MASC Cell line GBA mutation GD type 1 N370S/Y133* I 2 F213I/L444P III 3L444P/L444P III 4 IVS2 + 1G > A/N188S III The asterisk denotes aframeshift and consequential de novo stop codon

MASCs were induced to differentiate along the neuronal lineage at Day 0.At Day 1, cells were treated with mock (PBS) or 400 μM arimoclomol.Treatment persisted throughout differentiation for a total 9 days. AtDay 9, differentiation was evaluated by immunofluorescence of neuronalmarkers. GBA activity was measured using the fluorogenic substrate4-MUG.

Results

Arimoclomol does not Affect Neuronal Differentiation of Skin-DerivedHuman Multipotent Adult Stem Cells from GD TI and GDTIII Individuals

To assess the effect of arimoclomol on neuronal differentiation, MASCsfrom GD individuals were induced to differentiate while treated withmock or arimoclomol. Our results show that the expression of theneuronal markers Tubulin beta 3 and NeuN was not affected byarimoclomol.

Arimoclomol-Induced Increase in GBA Activity in Neurons from GD TI andGDTIII Individuals

We find that arimoclomol increases mutant GBA activity in neurons froman individual with GD type I (N370S/Y133*) and in three individuals withGD type Ill (F213I/L444P, L444P/L444P or IVS2+1G>A/N188S).

Taken together, our results demonstrate that arimoclomol increasesmutant GBA activity in neurons from GD TI and GD Till individualswithout affecting neuronal differentiation.

REFERENCES

-   Kieran, D., Kalmar, B., Dick, J. R. T., Riddoch-Contreras, J.,    Burnstock, G., & Greensmith, L. (2004). Treatment with arimoclomol,    a coinducer of heat shock proteins, delays disease progression in    ALS mice. Nature Medicine, 10(4), 402-405-   Mu, T., Ong, D. S. T., Wang, Y., Balch, W. E., Yates, J. R.,    Segatori, L., & Kelly, J. W. (2008). Chemical and biological    approaches synergize to ameliorate protein-folding diseases. Cell,    134(5), 769-81-   Bergamin, N., Dardis, A., Beltrami, A., Cesselli, D., Rigo, S.,    Zampieri, S., . . . Beltrami, C. A. (2013). A human neuronal model    of Niemann Pick C disease developed from stem cells isolated from    patient's skin. Orphanet Journal of Rare Diseases, 8(1), 34.-   Witte, M. D., Kallemeijn, W. W., Aten, J., Li, K.-Y., Strijland, A.,    Donker-Koopman, W. E., Aerts, J. M. F. G. (2010). Ultrasensitive in    situ visualization of active glucocerebrosidase molecules. Nature    Chemical Biology, 6(12), 907-13.

1. An active pharmaceutical ingredient selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of treating a glucocerebrosidase (GBA)-associated disorder.2. The active pharmaceutical ingredient for use according to claim 1,wherein said GBA-associated disorder is not Gaucher's disease (GD). 3.The active pharmaceutical ingredient for use according to claim 1,wherein said GBA-associated disorder is a GBA-associatedalpha-synucleinopathy.
 4. The active pharmaceutical ingredient for useaccording to any of the preceding claims, wherein said GBA-associatedalpha-synucleinopathy is selected from the group consisting ofGBA-associated Parkinson's disease (PD), GBA-associated dementia withLewi bodies (DLB) and GBA-associated multiple system atrophy (MSA). 5.The active pharmaceutical ingredient for use according to any of thepreceding claims, wherein said GBA-associated disorder is GBA-associatedparkinsonism.
 6. The active pharmaceutical ingredient for use accordingto any of the preceding claims, wherein said GBA-associated disorder isGBA-associated Parkinson's disease.
 7. The active pharmaceuticalingredient for use according to any of the preceding claims, whereinsaid GBA-associated disorder is associated with reduced GBA enzymelevels.
 8. The active pharmaceutical ingredient for use according to anyof the preceding claims, wherein said GBA-associated disorder isassociated with reduced GBA enzyme activity.
 9. The activepharmaceutical ingredient for use according to any of the precedingclaims, wherein said GBA-associated disorder is associated with reducedGBA enzyme activity and/or levels and said GBA gene is wild-type, andthe reduction in GBA activity is due to suppression of activity of theprotein or repression of transcription or translation of thegene/protein or is idiopathic.
 10. The active pharmaceutical ingredientfor use according to any of the preceding claims, wherein saidGBA-associated disorder is associated with one or more individual GBAgene mutations.
 11. The active pharmaceutical ingredient for useaccording to any of the preceding claims, wherein the individual with aGBA-associated disorder remain clinically unaffected re Gaucher'sdisease.
 12. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said GBA-associated disorder isassociated with one or more heterozygous GBA gene mutation.
 13. Theactive pharmaceutical ingredient for use according to any of thepreceding claims, wherein said GBA-associated disorder is associatedwith a homozygous GBA gene mutation, wherein said GBA-associateddisorder is not Gaucher's disease.
 14. The active pharmaceuticalingredient for use according to any of the preceding claims wherein saidone or more individual GBA gene mutations are mild (associated with GDtype I).
 15. The active pharmaceutical ingredient for use according toany of the preceding claims wherein said one or more individual GBA genemutations are severe (associated with GD type II and III).
 16. Theactive pharmaceutical ingredient for use according to any of thepreceding claims wherein said GBA gene mutation is selected from thegroup consisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S,N370S/1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*,F213I, N188S and IVS2+1G>A/N188S.
 17. The active pharmaceuticalingredient for use according to any of the preceding claims wherein saidGBA gene mutation is L444P.
 18. The active pharmaceutical ingredient foruse according to any of the preceding claims wherein said GBA genemutation is E326K.
 19. The active pharmaceutical ingredient for useaccording to any of the preceding claims wherein said GBA gene mutationis N370S.
 20. The active pharmaceutical ingredient for use according toany of the preceding claims wherein said GBA-associated disorder is aL444P, A456P, V460V heterozygote.
 21. The active pharmaceuticalingredient for use according to any of the preceding claims wherein saidGBA-associated disorder is a GBA mutation carrier, such as an obligatecarrier, such as a carrier which is clinically unaffected re. Gaucher'sdisease.
 22. The active pharmaceutical ingredient for use according toany of the preceding claims wherein said GBA-associated disorder is aclinically unaffected parent or sibling of a Gaucher's disease patient.23. The active pharmaceutical ingredient for use according to any of thepreceding claims, wherein said GBA-associated Parkinson's disease isassociated with reduced GBA enzyme levels and/or reduced GBA enzymeactivity.
 24. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said GBA-associated Parkinson'sdisease is associated with one or more GBA gene mutations.
 25. Theactive pharmaceutical ingredient for use according to any of thepreceding claims, wherein said GBA-associated Parkinson's disease isassociated with one or more heterozygous GBA gene mutations.
 26. Theactive pharmaceutical ingredient for use according to any of thepreceding claims, wherein said GBA-associated Parkinson's disease isassociated with one or more homozygous or compound heterozygous GBA genemutations.
 27. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said GBA-associated Parkinson'sdisease is associated with one or more GBA gene mutations selected fromthe group consisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S,N370S/1-BP ins 84G, V394L, A456P, V460V, C342G, G325R and P415R.
 28. Theactive pharmaceutical ingredient for use according to any of thepreceding claims, wherein said GBA-associated Parkinson's disease isassociated with a heterozygous or a homozygous N370S/N370S GBA genemutation.
 29. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said GBA-associated Parkinson'sdisease is associated with a genetically high-risk Parkinson's diseaseGBA genotype.
 30. The active pharmaceutical ingredient for use accordingto any of the preceding claims, wherein said GBA-associated Parkinson'sdisease is associated with idiopathic reduced GBA enzyme activity and/orlevels with no accompanying GBA gene mutations identified.
 31. Theactive pharmaceutical ingredient for use according to any of thepreceding claims, wherein said active pharmaceutical ingredient is theracemate ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.
 32. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said active pharmaceuticalingredient is an optically active stereoisomer ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.
 33. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said active pharmaceuticalingredient is an enantiomer ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.
 34. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said active pharmaceuticalingredient is selected from the group consisting of(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, and(−)-(S)—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.
 35. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said active pharmaceuticalingredient is an acid addition salt ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride.
 36. The active pharmaceutical ingredient for use according toany of the preceding claims, wherein said active pharmaceuticalingredient is selected from the group consisting ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate, andN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate.
 37. The active pharmaceutical ingredient for useaccording to any of the preceding claims, wherein said activepharmaceutical ingredient is selected from the group consisting of(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate;(−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride citrate;(+)-R—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate; and(−)-S—N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride maleate.
 38. The active pharmaceutical ingredient for useaccording to any of the preceding claims, wherein said treatment isprophylactic, curative or ameliorating.
 39. An active pharmaceuticalingredient selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of reducing risk in an individual of developing aglucocerebrosidase (GBA)-associated disorder other than Gaucher'sdisease, wherein said individual has reduced GBA level and/or reducedGBA activity.
 40. The active pharmaceutical ingredient for use accordingto claim 39, wherein said individual with reduced GBA level and/oractivity has one or more GBA gene mutation, such as a heterozygous GBAgene mutation or such as a homozygous GBA gene mutation.
 41. The activepharmaceutical ingredient for use according to any of claims 39-40,wherein said GBA-associated disorder is a GBA-associatedalpha-synucleinopathy, such as a GBA-associated alpha-synucleinopathyselected from the group consisting of GBA-associated parkinsonism,GBA-associated Parkinson's disease (PD), GBA-associated dementia withLewi bodies (DLB) and GBA-associated multiple system atrophy (MSA). 42.The active pharmaceutical ingredient for use according to claim 40,wherein said GBA-associated disorder is GBA-associated Parkinson'sdisease.
 43. The active pharmaceutical ingredient for use according toany of claims 39-42, wherein said individual has GBA activity and/orlevels of about 5 to 95% of hypothetical wild type activity and/orlevels, such as 5 to 10%, such as 10 to 20%, such as 20 to 30%, such as30 to 40%, such as 40 to 50%, such as 50 to 60%, such as 60 to 70%, suchas 70 to 80%, such as 80 to 90%, such as 90 to 95% of hypothetical wildtype activity and/or levels.
 44. An active pharmaceutical ingredientselected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof, for usein a method of one or more of a. increasing GBA activity, b. increasingGBA levels (or amount), c. increasing the amount of active mutant GBA,d. increasing the amount of active wild type GBA, e. enhancing foldingof ER-retained mutant GBA, f. increasing the amount ofprocessed/maturated GBA, g. increasing the amount of matured (post-ER)GBA, h. increasing the amount of matured GBA reaching the lysosomes,and/or i. reducing alpha-synuclein aggregation.
 45. A composition, suchas a pharmaceutical composition, comprising—separately or together—anactive pharmaceutical ingredient selected fromN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoylchloride, its stereoisomers and the acid addition salts thereof(arimoclomol); and one or more further active ingredients; for use inthe treatment of a glucocerebrosidase (GBA)-associated disorder otherthan Gaucher's disease (GD), including GBA-associatedalpha-synucleinopathies such as GBA-associated Parkinson's disease (PD),GBA-associated dementia with Lewi bodies (DLB) and GBA-associatedmultiple system atrophy (MSA).